We investigated the relationship between mammary cancer and the presence of specific endogenous and infectious mouse mammary tumor virus (MMTV) genetic elements in murine tissue. Using hybridization kinetics, restriction endonuclease analysis and the Southern blot technique we have studied the MMTV proviral content of tissues from mice with a low (C3H/StWi), moderate (BALB/cV), or high (GR) incidence of spontaneous mammary tumors. Our studies in C3H/StWi mice have shown that amplification of endogenous MMTV sequences or infection with an exogeneous virus does not appear to be involved in tumorigenesis. Analysis of mammary tumor DNA from BALB/cV demonstrated integrated MMTV specific DNA not found in normal organs from these mice. We have subsequently isolated and characterized a new MMTV variant from these mice. This new virus differs biochemically and immunologically from other MMTV isolates. In the high tumor incidence GR strain, we have identified a single endogenous GR provirus which segregates with the incidence of mammary cancer in crosses of inbred mice, and appears to be responsible for inducing the high incidence of mammary cancer seen in this strain.